Myosin light-chain kinase can be phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. Phosphorlation of myosin kinase decreases the affinity of this enzyme for the calcium-binding protein calmodulin by twenty fold. Dephosphorylation of myosin kinase by a phosphatase purified from smooth muscle reverses this effect and increases calmodulin binding to myosin kinase. The ability of cyclic AMP-dependent protein kinase to decrease the activity of myosin light chain kinase, provides a direct mechanism for cyclic AMP mediated smooth muscle relaxation.